The goal of this proposal is to understand the mechanisms by which DNA adenine methylase (DAM), leucine-responsive regulatory protein (Lrp), and PapI orchestrate the reversible switch between OFF and ON Pap pili expression states in uropathogenic Escherichia coli (UPEC). Since Pap pili are an essential virulence determinant of UPEC, this work has direct application to addressing the problem of urinary tract infections. This work will serve as a paradigm to understand how DNA methylation patterns control heritable gene expression states. The core switch involves PapI-dependent translocation of Lrp between pap promoter proximal sites 1,2,3 and distal sites 4,5,6. The methylation states of two GATC sites within the central pap sites 2 and 5 (GATCprox and GATCdist, respectively) control binding of Lrp and Lrp-PapI. Binding of Lrp to promoter proximal sites represses pap transcription whereas binding of Lrp to distal sites is essential for activation of pap transcription. The first aim is to determine how PapI and DAM control binding of Lrp to sites 1,2,3 and 4,5,6. The hypothesis that PapI enhances binding of Lrp to sites 2 and 5 by interacting with pap DNA sequences and Lrp in a ternary complex will be tested. The base-pair contacts between Lrp-PapI and sites 2 and 5 will be identified by missing contact, SELEX, and methylation analyses. Regulatory mutant pap DNA's which bind Lrp normally but are no longer PapI-responsive will be used to test the hypothesis that methylation of GATCprox facilitates OFF to ON switching by specifically blocking PapI enhancement of Lrp binding at sites 1,2,3. The second aim is to identify amino acids of Lrp that play important roles in responsiveness to PapI and DNA methylation, which will be accomplished by isolation of lrp mutants with altered responses to these factors and by a genetic suppressor approach using pap mutants isolated in Aim 1. Photocrosslinking studies are proposed to directly identify amino acids within Lrp that interact with sites 2 and 5, and to determine how these interactions are altered by GATC site methylation. Amino acids at the protein-protein binding interface of PapI and Lrp will be identified using yeast one-hybrid and beta-lactamase complementation analyses. The third aim is to analyze real-time in vivo dynamics of phase variation, which will include a test of the hypothesis that DNA replication is required for Pap phase switching. This will be carried out by monitoring Pap pili gene expression by fluorescence activated cell sorting in synchronized cells following induction of Pap I. Further analysis of the methylation states of the pap GATC sites in wild-type and regulator mutant pap operons following passage of the replication fork will be done to link in vitro studies with in vivo switch dynamics. These studies will also provide a detailed framework for understanding epigenetic regulatory mechanisms in other prokaryotes and eukaryotes.